This proposal focuses on two major goals: 1) characterization of axonal tubulin and microtubules; and 2) evaluation of the roles played by tubulin and axonal growth and regeneration. Tubulin is one of the major proteins of brain and knowledge about the properties of neuronal tubulin and microtubules is essential for understanding the roles played by tubulin in neuronal function. Axonal transport studies have shown that the bulk of axonal tubulin is cold insoluble and biochemically distinct from the tubulin in microtubules prepared by cycling from whole brain. The morphological correlate of cold insoluble tubulin was shown to be stable segments of microtubules. Such stable microtubule segments would affect the dynamics of the axonal cytoskeleton and could function as local regulators of microtubule polymerization in the axon. The plan of research will involve development of procedures for purification of cold insoluble tubulin from nervous tissue. Both axonal tubulin labeled by axonal transport and purified cold insoluble tubulin will be analyzed to determine the biochemical basis of cold stability in axonal microtubules. Particular emphasis will be placed on possible posttranslation modification and differences in microtubule associated proteins. The properties and biological function of cold stable microtubules will be evaluated by examination of the effects of insoluble tubulin on microtubule assembly in vitro. The amount, morphological form, and changes in cold insoluble tubulin in different neurons and regenerating neurites will be evauated biochemically and immunochemically. These studies will provide a better understanding of the roles that tubulin and microtubules play in the function of neurons as well as identifying molecular mechanisms for modulating the properties of neuronal tubulin.